1. Field of the Invention
The invent relates to aquatic subunit vaccine and in particular, to an aquatic subunit vaccine prepared based upon recombinant DNA technology by fusing a fish antigenic protein with the receptor binding motif and the translocation domain of an exotoxin A, as well as with the signal peptide of KDEL.
2. Description of the Prior Art
Infectious Pancreatic Necrosis (IPN) is a worldwide highly contagious fish disease. Infectious Pancreatic Necrosis Virus (IPNV), the pathogen of IPN, belongs to the family of Birnaviridae. The genome of IPNV consists of two segments of double-stranded RNA (dsRNA), segments A and B. The virion is a non-enveloped icosahedron particle. The length of the A segment gene is 3.2 Kb and codes predominantly for primary virus binding protein (VP2), secondary virus binding protein (VP3), and enzyme protein (VP4).
The major hosts of Infectious Pancreatic Necrosis Virus are salmon and trout. Other economical fishes and shells like eels, barracudas, cods, tilapias, pond loaches, milkfish, and clams, had be found to be infected by IPNV. IPNV affects primarily the young fish of salmon and trout in the initial feeding stage. The young fish in the weeks of initial feeding stage and later in the freshwater stage are vulnerable to IPNV infection. Nevertheless, in the mid 1980's, indications of IPNV disease outbreaks in post-smolt salmons held in seawater had also been reported. IPNV can be traced in the primary Atlantic salmon aquaculture countries such as Chile, America, and Norway. In Norway, IPN is one of the most serious contagious fish diseases in the salmon aquaculture industry. IPNV can cause a mortality of near 100% in freshwater stage of salmon. In post-smolt salmons, the mortality of IPNV may range from 10-20% up to 70%. Therefore, IPNV is a severe threat, ecologically and economically, to the aquaculture and sea-farming industries.
Vaccination against IPNV is the major control approach used in the salmon aquaculture industry. Conventional aquatic vaccines against IPNV includes mainly two types, the pathogen killed vaccine and the subunit vaccine. The development of pathogen-killed vaccine requires cell lines non-infected by other viruses. However, the fish cell lines are uncommon and the options are rare in addition to its difficulty in development as well as the high cost therefore. Subunit vaccine can be manipulated by genetic engineering technology. VP2 protein of IPNV expressed by E. coli or structure proteins of IPNV expressed by Insect Baculovirus are two common types of IPNV subunit vaccines that can induce the specific antibody responses in salmons. However, protective immunological effects offered by those approaches described above can not satisfy the standard required in the aquaculture industry.
There are several theories as to why a subunit vaccine against viruses can not induce a protective immunity. Concepts that have several proponents are as follow:                1) the antigen can not properly targeted to the cell that plays the key role in inducing immunity;        2) the antigen may be trapped in exogenous routes and not transported into cytoplasm of a target cell to associate with its endoplasmic reticulum; and        3) as a consequence of the foregoing, the antigen may not be presented to the effector cell in the correct context.        
Accordingly, there are a lot of drawbacks associated with conventional aquatic vaccines and improvements thereof have to be improved urgently.
In recent years, as rapid development of biotechnologies, genetic engineering approaches are utilized to modify and alter genes, thereby microorganisms originally harmful to human being or animals can be transformed into beneficial tools in medical and agricultural application. For example, Pseudomonas aeruginosa is one of typical examples among these organisms. When a patient with cystic fibrosis is infected by Pseudomonas aeruginosa may develop often chronic respiratory tract infection followed with chronic pneumonia. As cancer patients with immunodeficiency or patients severely burned are infected by Pseudomonas aeruginosa tend to turn into acute pneumonia or sepsis. The main factor in causing diseases to infected patients is the exotoxin A produced by Pseudomonas aeruginosa. 
The protein structure of exotoxin A consists of three major functional domains. Domain I of exotoxin A is a receptor-binding domain that is responsible for binding with α2-macroglobulin/LDL receptor on the plasma membrane of a mammalian cell to form an internalized ligand-receptor complex and then enters into the endosome of the cell through endocytosis. Where it will be localized and processed by proteases within the endosome. After enzymatic cleavage of exotoxin A in endosome, truncated protein fragments containing domain II (the translocation domain) and the toxic domain III are released and further translocated under the action of the translocation domain II from endosome into cytoplasm where it will bind with Golgi body and endoplasmic reticulum (ER) to form reticulum-Golgi network. Then, the third domain of truncated exotoxin A enables to inhibit protein synthesis in the cell, resulting in cell death.
Currently, the exotoxin A of P. aeruginosa is used for developing anti-cancer medicines. Immunotoxin produced by fusing specific antibodies with exotoxin A can be targeted to specific types of cells, such as cancer cells, by taking the advantages of specificity associated with the conjugated antibodies. It is expected that the protein synthesis pathway will be blocked in the targeted cells, thereby achieves the purpose of destroying the growth of the particular cell.
In view of various disadvantages derived from conventional aquatic subunit vaccine and the application of P. aeruginosa on the genetic engineering, the inventor of this application has devoted to improve it and after studying intensively for many years, provides a novel aquatic subunit vaccine and thus accomplish the invention.